To improve access to limiting nutrients, the vast majority of land plants forms arbuscular mycorrhizal (AM) symbioses with Glomeromycota fungi. We show here that AM-related GRAS transcription factors from different subgroups are upregulated during a time course of mycorrhization. Based on expression studies in mutants defective in arbuscule branching (ram1-1, with a deleted MtRam1 GRAS transcription factor gene) or in the formation of functional arbuscules (pt4-2, mutated in the phosphate transporter gene MtPt4), we demonstrate that the five AM-related GRAS transcription factor genes MtGras1, MtGras4, MtGras6, MtGras7, and MtRad1 can be differentiated by their dependency on MtRAM1 and MtPT4, indicating that the network of AM-related GRAS transcription factors consists of at least two regulatory modules. One module involves the MtRAM1- and MtPT4-independent transcription factor MtGRAS4 that activates MtGras7. Another module is controlled by the MtRAM1- and MtPT4-dependent transcription factor MtGRAS1. Genome-wide expression profiles of mycorrhized MtGras1 knockdown and ram1-1 roots differ substantially, indicating different targets. Although an MtGras1 knockdown reduces transcription of AM-related GRAS transcription factor genes including MtRam1 and MtGras7, MtGras1 overexpression alone is not sufficient to activate MtGras genes. MtGras1 knockdown roots display normal fungal colonization, with a trend towards the formation of smaller arbuscules.

Chlorinated ethene contamination is a worldwide relevant health issue. In anaerobic aquifers, highly chlorinated ethenes are transformed by microbially-mediated organohalide respiration metabolism. For this reason, in the last few years, bioremediation interventions have been developed and employed in situ for aquifer decontamination. Biostimulation has been demonstrated to be efficient in enhancing organohalide respiration activity. The use of agrifood wastes that replace engineered substrates as biostimulants permits the low carbon impact of bioremediation treatment as part of a circular economy approach. The present work depicts the effects of available bio-based substrates and discusses their efficiency and impact on microbial communities when applied to contaminated aquifers. As a drawback of anaerobic organohalide respiration, there is the accumulation of more toxic lower-chlorinated ethenes. However, compounds such as dichloroethene (DCE) and vinyl chloride (VC) can be mineralized by metabolic and co-metabolic pathways in aerobic conditions. For this reason, sequential anaerobic/aerobic treatments proposed to stimulate the natural biotransformation activity can achieve complete degradation of chlorinated ethenes. The aim of this work is to provide an up-to-date revision of anaerobic/aerobic microbial transformation pathways towards chlorinated ethenes and to discuss their application in real scenarios and futurable microbial bioelectrochemical systems to remediate contaminated aquifers.

In this study, a bioremediation approach was evaluated for the decontamination of an aquifer affected by the release of organohalides by an industrial landfill. After preliminary physicochemical and microbiological characterization of the landfill groundwater, the stimulation of natural organohalide respiration by the addition of a reducing substrate (i.e., molasse) was tested both at microcosm and at field scales, by the placement of an anaerobic permeable reactive bio-barrier. Illumina sequencing of cDNA 16S rRNA gene revealed that organohalide-respiring bacteria of genera Geobacter, Sulfurospirillum, Dehalococcoides, Clostridium and Shewanella were present within the aquifer microbial community, along with fermentative Firmicutes and Parvarchaeota. Microcosm experiments confirmed the presence of an active natural attenuation, which was boosted by the addition of the reducing substrate. Field tests showed that the bio-barrier decreased the concentration of chloroethenes at a rate of 23.74 kg d⁻¹. Monitoring of organohalide respiration biomarkers by qPCR and Illumina sequencing revealed that native microbial populations were involved in the dechlorination process, although their specific role still needs to be clarified. The accumulation of lower-chloroethenes suggested the need of future improvement of the present approach by supporting bacterial vinyl-chloride oxidation, to achieve a complete degradation of chloroethenes.

In situ bioremediation (ISB) is a widely accepted method for eradicating petroleum hydrocarbons (PHCs) and chlorinated aliphatic hydrocarbons (CAHs) from contaminated aquifers. To achieve full removal of all toxic compounds that originated from microbial degradation, sequential anaerobic/aerobic bioremediation systems are recommended. While several works based on laboratory analyses targeting sequential bioremediation have been documented, examples of sequential ISB are limited. The purpose of this study is to report and analyze the results obtained from the multiscale characterization activities propaedeutic to the construction of Italy’s largest (> 400 m long) sequential ISB system. The rich wealth of information produced during this study provides a useful example that can be followed for the construction of new sequential ISBs. The system was set up to remediate a solute plume containing PHCs and CAHs in an alluvial aquifer in Italy. Microcosm experiments were carried out to determine the biodegradation potential under anaerobic and aerobic conditions. In situ tests were performed by installing two 40-m-long pilot biobarriers for sequential anaerobic and aerobic degradation intercepting part of the contamination plume. These experiments pointed out the need of adding biostimulating compounds to accelerate the biodegradation process, under both aerobic and anaerobic conditions. In situ tests showed removal efficiencies of up to 95 and 99% for total CAHs and PHCs, respectively, proving the feasibility of the full-scale ISB system. Apparent discrepancies between laboratory and in situ tests can be ascribed to scale effects and aquifer heterogeneities.